A Study About The Expression And Regulation Factors Of LncRNA MEG3 And H19 In The Pig Skeletal Muscle

Skeletal muscles are attached to the bone,a type of striated muscle whose main function is the exercise movement.The distribution of blood vessels and lymphatic organization around is very rich.Due to innervation,so it is also known as the "voluntary muscle".Skeletal muscle fibers generally have multiple nuclei.There is a layer of muscle satellite cells between muscle fibers and basal membrane,the cells that have the ability to repair damaged muscle fibers.Body movement is mainly done by the contraction of skeletal muscles,always with the process of glucose decomposition and energy release.Daniel Bovet think that skeletal muscle can play an important role in the use of blood glucose.In the human body,the vast majority of glucose eventually complete the conversion and storage in the skeletal muscle.Patients with diabetes are generally associated with the coming decay or atrophy of skeletal muscle.The damaged skeletal muscle cells can also trigger rhabdomyolysis(RM),furthermore them may lead to acute renal failure(ARF).So the research of skeletal muscle could be used to understand the pathogenesis of skeletal muscle related diseases.Daily consumption of meat in China mainly comes from the pigs.The nutrition and flavor of pork mainly depends on the content of muscle and fat.Lean meat is an important measure of pig muscle content,and the pig muscle is generally made up of skeletal muscle.Pig skeletal muscle consists of dorsal muscle and leg muscle.Researches on the growth and development of pig skeletal muscle have a great significance meaning in the pig genetic breeding improvement.The study of the function and regulation of LncRNA MEG3 and H19 in the pig skeletal muscle development is unclear,therefore this article undertakes identification of the core promoter and interactive verification of the related miRNA of MEG3 gene,the construction of the organization and time-spectrum expression and SNPs classification parting correlation analysis of both genes,in order to make the foundation for further research on the regulation mechanism of LncRNA MEG3 and H19 gene involved in skeletal muscle growth.1.MEG3 core promoter gene identificationBy cloning landrace MEG3 gene promoter and look for its core function area,forecasting transcription factors combined with MEG3 gene promoter,we offer a foundation to clarify the specific transcription regulation mechanism in skeletal muscle.Landrace blood genomic DNA as a template,the polymerase chain reaction(PCR)is used to obtain MEG3 gene 5 ’flanking sequence.According to the determination of nucleic acid sequencing,the results of clone to MEG3 gene 5’ flanking sequence match the record ones in GenBank.Then it was inserted into pGL3-Basic carrier.Using dual luciferase reporter gene detection system,we identify its promoter activity.Luciferase activity analysis indicates that in pigs PIEC and PK15 cells the segment has a strong promoter activity.By means of gradual loss analysis respectively from the 5’ flank segment of MEG3,in which the different truncated cloning segments inserted pGL3-Basic carrier and used dual luciferase reporter gene detection system identified those promoter activity for recognizing core function area.Results show that the activity in 5 ’end of missing truncated segments compared with pGL3-Basic is first reduced after rising at-440～+ 86 bp cut short fragment whose activity closes to the top,and-40～+ 86 bp truncated segment activity almost none.That prompt MEG3 core promoter regions may between-447～-40 bp and there may be a core control element in the area.According to bioinformatics analysis,the area contains transcription factors binding sequence,including MyoD,SP1,NFY,is binding sites and E47.2.MEG3 mirna target validation associated with skeletal muscle growth and developmentBy cloning MEG3 part fragment,predict and verify its interactions with miRNAs,we try to clarify the regulatory mechanism of MEG3 at the transcription level involved in the process of skeletal muscle growth and development.Landrace skeletal muscle cell RNA as template,by the method of combining RT-PCR and touch down PCR,we cloned MEG3 part sequence.After the determination of nucleic acid sequencing,the results of clone to MEG3 part of the sequence is consistent with the record in GenBank,then insert the psi-Check2 carrier.Through bioinformatics method we predicted potential microRNA interacted with MEG3 clone sequence at the same time.Bioinformatics analysis results showed that the area contains miR133a,miR1 33b and miR378 potential binding sites.Then we used the double luciferase reporter gene detection system for target validation.Luciferase activity analysis results show that the proposed miR133a in pig PK15 and PIEC cells,the role of MEG3 sequence was not significant,while the miR133b and miR378 combined with MEG3 sequence has strong activity.By using the method of RT-qPCR we detected the expression of mircoRNA with MEG3 in spectrum and tissue,real-time quantitative PCR results showed that the relative expression of MEG3 in landrace skeletal muscle and kidney tissue increased,and in the process of skeletal muscle growth and development,focused on the embryonic period,its expression reached the maximum in embryonic period of 60 days.And by trends related analysis of both embryonic period,the results show that the miR378 have a significant correlation with MEG3 in expression trends.Those prompt MEG3 may interact with miR378.miR378 may play a control role in MEG3 potential interactive mirna.3.The large white MEG3 cloning of gene sequence and polymorphism,haplotype and back fat growth properties such as relevanceBy cloning large white MEG3 gene coding sequence and looking for potential SNP loci,we did correlation analysis between genotypes including the linkage disequilibrium haplotype and SNPs with the traits related to the development of skeletal muscle growth,such as backfat correction and corrective age and loin eye area up to 100 kg,in order to provide theoretical basis for the further study of skeletal muscle growth and development.According to swine reported MEG3 gene sequences,we cloned pigs MEG3 gene fragment sequence,then using DNAStar software of SeqMan to sequence splicing,finally we get a total of 5234 bp of DNA sequences including 2 exons and introns 1.according to the results of determination of nucleic acid sequence,cloning MEG3 gene sequences are basically identical with GenBank records.And then we designed the primers and used 32 pig blood mixed DNA pools over the method of segmented amplification,and then direct sequencing.In mixing pool sequencing map,we get 11 potential SNP loci with existence of bimodal.Through looking for the RS in the NCBI for 11 SNP annotation,we found that eight SNPS has the known RS,including rs322438324,rs318656749,rs81286029.rs344501106,rs325797437,rs334059356.rs323571592,rs322802425 and three new SNPS including rs25,rs24,rs23.Eventually we sequencing 297 large white by mass spectrometry,mass spectrometry analysis results show that except the rs24 testing out,other sites were successfully detected genotype.And then we used the Haploview,GraphPad and R language rectify the sequencing results and did statistical correlation analysis between genotypes including the linkage disequilibrium haplotype and SNPs with the properties related to the development of skeletal muscle growth.Haploview analysis shows that only rs334059356,rs325797437,rs344501106,rs81286029,rs318656749,rs25,rs322438324 have a total alleles type;Haplotype analysis results show that there are three kinds of haplotypes of linkage disequilibrium.PCA results show that for the same character,the same gene type and haplotypes tend to gather together.And correlation analysis result shows that in corrected backfat between TT and CT(p=0.0240<0.05)on rs318656749、AA and AG(p=0.0487<0.05)on rs81286029、AA and AG(p=0.0471<0.05)on rs344501106,there were significant differences.In the age of reaching 100 kg between AG and GG(p =0.0341<0.05)on rs325797437,AT and AA(p = 0.0357<0.05)on rs334059356,there were significant differences.Among the rest of the SNP classification there is no significant difference.Among haplotypes,there are significant difference beween AAAT homozygote and GAAT/GGGC heterozygote(p=0.0301<0.05)、two heterozygote AAAT/GGGC and GAAT/GGGC(p=0.0184<0.05)in the age of reaching 100 kg.Finally using the RNADraw drawed LncRNA MEG3 secondary structure with SNP mutant loci which have different phenotype of,we found rs325797437 locus mutation may lead to the change of secondary structure.That suggest possibly rs325797437 by changing the secondary structure of MEG3 transcription may play different biological effects,then influence the individual phenotype.4.The large white area H19 gene coding sequence of clone and its polymorphism,haplotype and back fat growth properties such as relevanceBy cloning large white H19 gene coding sequence and looking for potential SNP loci,we did correlation analysis between genotypes including the linkage disequilibrium haplotype and SNPs with the traits related to the development of skeletal muscle growth,such as backfat correction and corrective age and loin eye area up to 100 kg,in order to provide theoretical basis for the further study of skeletal muscle growth and development.This study based on NCBI pig H19 gene coding sequence,cloning distinguish swine H19 gene coding sequence,using DNAStar software of SeqMan sequence splicing,ultimately including five 6 exons and introns,a total of 3352 bp of DNA sequences.According to the results of determination of nucleic acid sequence,cloning H19 gene sequences are basically identical in GenBank records.And then we designed the primers and used 32 pig blood mixed DNA pools over the method of segmented amplification,and then direct sequencing.In mixing pool sequencing map,seven potential SNP loci exist bimodal.Then we marked the annotation of seven SNPs,including RS15,RS16,RS17,RS18,RS20,RS21,RS22.Finally we used mass spectrometry to sequencing 297 large white samples,and then we used the Haploview,GraphPad and R language rectify the sequencing results and did statistical correlation analysis between genotypes including the linkage disequilibrium haplotype and SNPs with the properties related to the development of skeletal muscle growth.Haploview analysis shows that seven sites all have allele type and there are three area of linkage disequilibrium.PCA results show that for the same character,the same genotype tend to gather together.And correlation analysis results of properties show that in the age of reaching 100 kg between TT and CT on rsl5(p = 0.0462<0.05)there were significant differences.But the genotypes of the rest SNPs does not exist significant differences.In the 100 kg corrective eye muscle area,between CC and CT(p = 0.0374<0.05)as well as TT and CT on rs 20(p = 0.0478<0.05)were significantly difference,especially between CC and TT shows extremely significant differences(p = 0.0093<0.01).To further correlation analysis of haplotype,we found in the haplotype of RS15 and RS16,TcCt and CcCt(p = 0.0498<0.05)are significant differences in corrective backfat;In the haplotype of RS17 and RS18,CcTt and CtTg(p = 0.0371<0.05)in the correct eye muscle area are significant differences.Using qRT-PCR to detect the expression level of H19 in nine tissue and 15 period of skeletal muscle growth and development,expression spectrum analysis found that H19 is mainly expressed in skeletal muscle and kidney and furthermore in an early stage in skeletal muscle development it showed high specificity expression on embryonic 105 days.Those prompt H19 gene exists SNP loci associated with skeletal muscle growth traits and is likely to be.different expressed genes in skeletal muscle.